An object of the invention is to provide a process for the synthesis of modified P-chiral nucleotide analogues of general formula 1, where R1 stands for protecting group, preferably 4,4xe2x80x2-dimethoxytrityl (DMT), 9-phenylxanthene-9-ol (Px) or trialkylsilyl group, R2 is a hydrogen atom, protected hydroxyl group, halogen, chloroalkyl, nitrile, azide, protected amine, perfluoroalkyl (containing up to four carbon atoms), perfluoroalkoxyl (containing up to four carbon atoms and up to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl, OQ1, SQ1, NHQ1, where Q1 stands for alkyl (C1-C4), aryl (C6-C12), alkenyl (C3-C12) or alkynyl (C3-C12), B stands for a purine or pyrimidine base (appropriately protected if necessary), Z is selected from Q1 or vinyl, ethynyl, aminomethyl or aminoethyl substituents, X means oxygen, sulfur or selenium atom, Rx is a protecting group, preferably aroyl, acyl, alkoxycarbonyl, benzenesulfonic, alkyl, trialkylsilyl group or the next unit of elongated oligonucleotide chain.
Bacterial or viral infection, as well as uncontrolled proliferation of cancer cells in a living organism, lead to a fully developed disease predominantly by synthesis of xe2x80x9cunwantedxe2x80x9d, harmful proteins. Viral diseases result from incorporation of viral genetic information into a host""s genome followed by synthesis of viral proteins, which are damaging to the host organism.
Caused by different factors aberrations of protooncogenes and formation of oncogenes responsible for synthesis of xe2x80x9cunwantedxe2x80x9d proteins are recognized as important factors in cancer cells proliferation processes.
Recent achievements in molecular biology, including explanation of molecular bases of such diseases as AIDS, different viral and cancer diseases or blood circulation disesaes, resulted in intensive search for new selective treatments aimed at inhibition of the expression of genes which code xe2x80x9cunwantedxe2x80x9d proteins, or at tuning of the level of known regulatory proteins.
Two newly developed therapeutic approaches are ANTISENSE mRNA (C. A. Stein, Cancer Res., 1988, 48, 2659) and ANTIGENE (N. T. Thuong et al., Angew.Chem.Int.Ed.Engl., 1993, 32) strategies, which stem from the knowledge on interactions between oligo(deoxyribonucleotide)s and DNA or RNA molecules. These conceptions are based on the assumption that short synthetic oligo(deoxyribonucleotide)s after being delivered inside a cell, form stable duplexes with complementary DNA or RNA molecules, and on this way slow down either transcription or translation process (E. Wickstrom, ed. Wiley-Liss, New York N.Y. 1993, xe2x80x9cProspects for Antisense Nucleic Acid Therapy for Cancer and AIDSxe2x80x9d).
Nucleolytic enzymes present in cells and body fluids are able to hydrolyze exogenous DNA molecules very rapidly, thus stability of oligo(deoxyribonucleotide)s and their analogues against nucleases is a crucial factor in respect to their in vivo activity. Majority of modifications introduced to the oligo(deoxyribonucleotide)s with the aim of their enhanced nucleolytic stability, involved changes of ligands attached to the phosphorus atom of the internucleotide phosphodiester bond. Among them phosphorothioate, methanephosphonate, phosphoramidate and triester analogues to various extent fulfill the criterion of full or, at least, significantly enhanced stability. However, such modifications usually result in reduced hybridization properties towards complementary DNA and RNA strands (J. S. Cohen, ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla., 1989).
Applicability of antisense oligonucleotides as potential therapeutics depends upon their ability to cross the cellular membranes to reach necessary therapeutic concentration at the site of target molecules inside the cell (e.g. mRNA in cytoplasm). The cellular membranes made of protein-lipid layers are permeable only for small non-ionic molecules and are not permeable for most of natural metabolites and many drugs.
Natural and modified oligonucleotides complementary to fragments of viral DNA (RNA) are reported to show antiviral and anticancer properties in cell lines (in vivo), thus they are able to permeate through cell membranes and hybridize to the target DNA or RNA molecules. Several nucleolytically stable DNA analogues, as alkyl triesters (P. S. Miller, Biochemistry, 1977, 16, 1988), and methanephosphonates (C. H. Marcus-Sekura et al., Nucleic Acids Res., 1987, 15, 5749; P. S. Miller et al., Biochemistry, 1986, 25, 5092; S. K. Loke et al., Top. Microbiol. Immunol., 1988, 141, 282; A. M. Tari et al., J.Biol.Med., 1996, 74, 623; S. Agrawal et al., Clin.Pharmacokinet., 1995, 28, 7) were used for the research in different cell lines including human HL60, Syrian hamster fibroblasts, U 937, L 929, CV-1 and ATH 8. For modified oligonucleotides the cellular uptake is usually rather low, what results in reduced in vivo activity compared to that expected from in vitro studies.
So far, DNA analogues have worse hybridization properties than natural DNA, thus the inhibition of transcription or translation, and, consequently, inhibition of protein biosynthesis are less effective than expected. There are several reasons for this phenomenon, such as complicated third-order structure of RNA, limited accessibility of its particular segments, or DNA/RNA interactions with proteins.
In order to overcome these obstacles several DNA analogues possessing internucleotide linkages without phosphorus atom, like methylene group (M. Matteuci, Tetrahedron Lett., 1990, 31, 2385) dialkylsilyl groups (R. Stirczak, J.Org.Chem., 1987, 52, 202) or sulfonyl group (S. Benner, J.Org.Chem., 1995, 61, 7620) have been synthesized. Research on their application as therapeutics is in an initial phase, mostly because of unfavorable physicochemical properties, as poor solubility and hybridization properties, and low chemical stability. Triester analogues are degradable by esterases, what renders them unusable in the antisense strategy (Goodrick et al., Bioconj.Chem., 1990, 1, 165).
In the case of phosphorothioate and methanephosphonate analogues of DNA, which possess chiral center at the phosphorus atom, an additional problem is encountered, since the synthesis of oligomers with n internucleotide bonds results in formation of 2n diastereoisomers, unless the method of synthesis is stereospecific.
It was found, that for oligo(nucleoside-3xe2x80x2,5xe2x80x2-methanephosphonate)s of RP-, SP- or random configuration at each phosphorus atom, their hybridization properties towards complementary DNA or RNA depend on the configuration of the phosphorus centers (P. S. Miller et al., J.Biol.Chem., 1980, 255, 9659; Biochemistry, 1982, 21, 2507). For phosphorothioate DNA analogues the stereodifferentiation of hybridization properties is accompanied by their stereoselective susceptibility to enzymatic hydrolysis by certain nucleases (Potter et al., Biochemistry, 1983, 22, 1369; Bryant et al., Biochemistry, 1979, 18, 2825).
Lexc5x9nikowski et al.(Nucleic Acids Res., 1990, 18, 2109) found that stereospecifically synthesized octamer possessing six out of seven internucleotide methanephosphonate bonds of RP configuration has much stronger affinity towards pentadeoxyadenylic template than its counterpart possessing these bonds of SP configuration, or the oligomer obtained by non stereoselective method. The stereoregular oligomers were obtained by non stereoselective condensation of corresponding two stereoregular tetramers synthesized in solution starting from diastereomerically pure 5xe2x80x2-O-MMT-thymidine-3xe2x80x2-O-(O-p-nitrophenylmethanephosphonate)s and 3xe2x80x2-O-acetylthymidine with Grignard reagent used as an activator (Lexc5x9nikowski et al., Nucleic Acids Res., 1990, 18, 2109; ibid, 1988, 16, 11675; Lexc5x9nikowski et al., Nucleosides and Nucleotides, 1991, 10, 773).
Other examples of synthesis of diastereomerically pure (or, at least, significantly enriched with an RP diastereoisomer) methanephosphonate analogues of DNA involve reactions of methyidichlorophosphine with appropriately protected at the 5xe2x80x2 (first step) and 3xe2x80x2 (second step) nucleosides, carried out at low temperature (xe2x88x9280xc2x0 C.) in the presence of amines (including chiral amines). The highest obtained ratio of RP to SP isomers was 8:1 (Loscher, Tetrahedron Lett., 1989, 30, 5587; Engels et al., Nucleosides and Nucleotides, 1991, 10, 347) This method allows to synthesize dinucleoside methanephosphonates in diastereoselective manner.
Another method for stereoselective formation of internucleotide methanephosphonate bond is a reaction employing separated diastereoisomers of 5xe2x80x2-O-DMT-N-protected nucleoside 3xe2x80x2-O-(Se-alkylmethanephosphonate)s and appropriate 3xe2x80x2-5xe2x80x2-OH-(N-protected) nucleosides in the presence of DBU and lithium chloride (Woźniak et al.,J.Org.Chem., 1994, 58, 5061).
Recently, numerous laboratories have paid efforts to implement as therapeutics so called xe2x80x9cchimericxe2x80x9d oligomers, possessing phosphate or phosphorothioate xe2x80x9ccorexe2x80x9d flanked at both 5xe2x80x2 and 3xe2x80x2 ends by methanephosphonate units of RP configuration. The chimeras have enhanced stability against nucleases due to the presence of enzymatically stable internucleotide methanephosphonate linkages. Incorporation of methanephosphonate units only of RP configuration results in enhanced hybridization properties of the xe2x80x9cchimericxe2x80x9d product (M. Reynolds et al., Nucleic Acids Res., 1996, 24, 4584).
A process for the synthesis of modified P-chiral nucleotide analogues of general formula 1, where:
R1 stands for protecting group, preferably 4,4xe2x80x2-dimethoxytrityl (DMT), 9-phenylxanthene-9-ol (Px) or trialkylsilyl group,
R2 is a hydrogen atom, protected hydroxyl group, halogen, chloroalkyl, nitrile, azide, protected amine, perfluoroalkyl (containing up to four carbon atoms), perfluoroalkoxyl (containing up to four carbon atoms and up to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl, OQ1, SQ1, NHQ1, where Q1 stands for alkyl (C1-C4), aryl (C6-C12), alkenyl (C3-C12) or alkynyl (C3-C12),
B stands for a purine or pyrimidine base (appropriately protected if necessary),
Z is selected from Q1 or vinyl, ethynyl, aminomethyl or aminoethyl substituents,
X means oxygen, sulfur or selenium atom,
and Rx is a protecting group, preferably aroyl, acyl, alkoxycarbonyl, benzenesulfonic, alkyl, trialkylsilyl group or the next unit of elongated oligonucleotide chain
according to the present invention, consists in reaction of compound of formula 2, where R1, R2, B and Z have the above mentioned meanings, while Y stands for XR3 substituent, where X means oxygen, sulfur or selenium atom, and R3 means acyl group of formula COR4, in which R4 stands for alkyl (up to six carbon atoms), perfluoroalkyl (containing up to four carbon atoms), aroyl (containing six up to fifteen carbon atoms), preferably mono-, di- or trisubstituted aromatic substituents (xe2x80x94C6H4R5, xe2x80x94C6H3(R5)2 orxe2x80x94C6H2(R5)3, respectively), where R5 means a hydrogen atom, methyl substituent, halogen atom or other substituent activating the aromatic ring, with compound of formula 6, where B, R2 and Rx have the above mentioned meanings, under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield compound of formula 1, which then is isolated, and if X means a sulfur or selenium atom compound of formula 1 is oxidized with known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, followed by isolation of resulting 1 (where X means an oxygen atom and R1, R2, Rx, B and Z have the above mentioned meanings) using known methods. The process according to the present invention is carried out preferably in tetrahydrofuran or acetonitrile.
As an activating reagent in the reaction between compounds of formula 2 and formula 6 one can use organic bases, preferably amines, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).
In the process according to the present invention it is preferred to use an additional activator selected from a group consisting of lithium salts, especially lithium halides.
Another variant of the process for the synthesis of modified P-chiral nucleotide analogues of general formula 1 in the form of pure diastereomer of preselected configuration at the P-atom, where R1, R2, Rx, B, X and Z have the above mentioned meanings, according to the present invention consist in reaction of one of two diastereomers of compound of formula 2 of the configuration at the P-atom identical to that desired in the product, where R1, R2, B and X have the above mentioned meanings, while
Y stands for XR3 substituent, where X means an oxygen, sulfur or selenium atom, R3 means acyl group of formula COR4, in which R4 stands for alkyl (up to six carbon atoms), perfluoroalkyl (containing up to four carbon atoms), aryl (containing six up to fifteen carbon atoms), including mono-, di- or tri-substituted aromatic substituents (xe2x80x94C6H4R5, xe2x80x94C6H3(R5)2 or xe2x80x94C6H2(R5)3, respectively), where R5 means a hydrogen atom, methyl substituent, halogen atom or other substituent activating the aromatic ring,
with compound of formula 6, where B, R2 and Rx have the above mentioned meanings, under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield compound of formula 1, which then is isolated, or, if X means a sulfur or selenium atom, compound of formula 1 is oxidized with known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, followed by isolation of resulting 1 (where X means an oxygen atom and R1, R2,Rx, B and Z have the above mentioned meanings) using known methods.
The process according to the present invention is carried out preferably in tetrahydrofuran or acetonitrile. As the activating reagent in the reaction between compounds of formula 2 and formula 6 one can use organic bases, preferably amines, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), and as an additional activator compounds selected from a group consisting of lithium salts, especially lithium halides, can be used.
The third variant of the process for the synthesis of modified P-chiral nucleotide analogues of general formula 1 in the form of pure diastereomer of preselected configuration at the P-atom, where R1, R2, Rx, B, X and Z have the above mentioned meanings, according to the present invention consist in hydrolysis of one of two diastereomers of compound of formula 2 of the configuration at the P-atom opposite to that desired in the product of formula 1, while in the formula 2 R1, R2, B, Z, X and Y have the above mentioned meanings, in the presence of activator being able to invert an absolute configuration of the P-atom, while resulting product of general formula 2, where R1, R2, and B have the above mentioned meanings, while Y stands for an oxygen atom and X means a sulfur or selenium atom, is reacted with compound of general formula 7, where R4 stands for alkyl (up to six carbon atoms), perfluoroalkyl (containing up to four carbon atoms), aroyl (containing six up to fifteen carbon atoms), including mono-, di- or tri-substituted aromatic substituents (xe2x80x94C6H4R5, xe2x80x94C6H3(R5)2 or xe2x80x94C6H2(R5)3, respectively), where R5 means a hydrogen atom, methyl substituent, halogen atom or any other substituent, and W means a chlorine, bromine or iodine atom, to yield compound of formula 2, where R1, R2, B and Z have the above mentioned meanings, X means a sulfur or selenium atom, while Y stands for R4C(O)Oxe2x80x94, in which R4 has the above mentioned meaning, possessing the absolute configuration at the P-atom opposite to that in the starting material, and identical to that required for the product of formula 1, further possibly combined with the same diastereoisomer of formula 2 obtained from the earlier separation, and then reacted with compound of formula 6, where B, R2 and Rx have the above mentioned meanings, under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield compound of formula 1 of desired absolute configuration at the P-atom, which then is isolated, or, if X means a sulfur or selenium atom, compound of formula 1 is oxidized with known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, followed by isolation of resulting 1 (where X means an oxygen atom and R1, R2, Rx, B and Z have the above mentioned meanings) using known methods. The process according to the present invention is carried out preferably in tetrahydrofuran or acetonitrile.
As an activating reagent in the reaction between compounds of formula 2 and formula 6 one can use organic bases, preferably amines, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and as an additional activator lithium salts, especially lithium halides, are used.
The fourth variant of the process for the synthesis of modified P-chiral nucleotide analogues of general formula 1 in the form of pure diastereomer of preselected configuration at the P-atom, where R1, R2, Rx, B and Z have the above mentioned meanings, according to the present invention consist in reaction of one of two diastereomers of formula 2 of the configuration at the P-atom opposite to that desired in the product 1, while in the formula 2 R1, R2, B, Z, X and Y have the above mentioned meanings, with alcohol, preferably with methanol, possibly in the presence of activator, while the resulting product of general formula 2, where R1, R2, Z and B have the above mentioned meanings, while Y stands for an alkoxyl group, preferably methoxyl, and X means a sulfur or selenium atom, is further dealkylated using amines, preferably trimethylamine or t-butylamine, and the resulting compound of formula 2, where R1, R2, Z and B have the above mentioned meanings, while Y stands for an oxygen atom and X means a sulfur or selenium atom, is subsequently reacted with compound of general formula 7, where R4 and W have the above mentioned meanings, to yield compound of formula 2, where R1, R2, Z and B have the above mentioned meanings, X means a sulfur or selenium atom, while Y stands for R4C(O)Oxe2x80x94, possessing the absolute configuration at the P-atom opposite to that in the starting material, and identical to that required for the product of formula 1, further possibly combined with the same diastereoisomer of formula 2 obtained from the earlier separation, and then reacted with compound of formula 6, where B, R2 and Rx have the above mentioned meanings, under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield compound of formula 1 of desired absolute configuration at the P-atom, which then is isolated, or, if X means a sulfur or selenium atom, compound of formula 1 is oxidized with known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, followed by isolation of resulting 1 (where X means an oxygen atom and R1, R2, Rx, B and Z have the above mentioned meanings) using known methods. The process according to the present invention is carried out preferably in tetrahydrofuran or acetonitrile.
As an activating reagent in the solvolysis and in the reaction between compounds of formula 2 and formula 6 one can use organic bases, preferably amines, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and as an additional activator lithium salts, especially lithium halides, are used.
In the process according to the present invention preferably used compounds are those of general formula 2, obtained by phosphorylation of corresponding substrates of general formula 3, where R1, R2 and B have the above mentioned meanings, with phosphorylating reagents of general formula 4, where Z and X have the above mentioned meanings, W means a halogen atom, preferably chlorine, followed by hydrolysis without isolation of the intermediate 5, to yield compounds of formula 2, where R1, R2, B, Z and X have the above mentioned meanings, and Y means an oxygen atom.
Using the first variant of the process according to the present invention, pure diastereoisomers of formula 2 are transformed separately to yield pure diastereoisomers of compound 1.
More useful variant of the process according to the present invention consist in the reaction of compound of formula 3, where R1 and B have the above mentioned meanings, with compound of formula 4, where X means a sulfur or selenium atom and Z has above mentioned meanings, to yield compound of formula 5, where X means an oxygen, sulfur or selenium atom, which is then hydrolyzed to yield compound of formula 2 where R1, R2, Z and X have the above mentioned meanings and Y means an oxygen atom, and separated chromatographically into two diastereomers, followed by reaction with compound of formula 7, where R4 stands for alkyl (up to six carbon atoms), perfluoroalkyl (containing up to four carbon atoms), aroyl (containing six up to fifteen carbon atoms), including mono-, di- or tri-substituted aromatic substituents (xe2x80x94C6H4R5, xe2x80x94C6H3(R5)2 or xe2x80x94C6H2(R5)3, respectively), where R5 means a hydrogen atom, methyl substituent, halogen atom or any other substituent activating an aromatic ring, and W means a halogen, preferably chlorine. One isomer is reacted with compound of formula 6, to yield stereospecifically compound of formula 2, where Y stands for R4C(O)Oxe2x80x94, in which R4 has the above mentioned meaning. This isomer of 2 is reacted with compound of formula 6, where B, R2 and Rx have the above mentioned meanings, in the presence of an activating reagent as an organic base, preferably tertiary amine, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Diastereomerically pure compound of formula 1 is isolated using known methods. In the process according to the present invention the product 1 is oxidized with known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, to yield product 1, where X means an oxygen atom and R1, R2, Rx, B and Z have the above mentioned meanings.
In this variant the second diastereoisomer of 2 (Y=R4C(O)Oxe2x80x94) is reacted with alcohol (preferably methanol), and without isolation of intermediary 2, where R1, R2, Z and B have the above mentioned meanings, while Y stands for an alkoxyl group, preferably methoxyl, and X means a sulfur or selenium atom, is further dealkylated using strong base, preferably organic base, most preferably amine. This diastereoisomer has an absolute configuration opposite to that of the substrate 2, thus within the described above process inversion of configuration at the P-atom in compound of formula 2 takes place. The described variant of the invention allows to obtain compound of formula 2 (Z=O, X=S, Se) in which absolute configuration at the phosphorus atom is 100% inverted, starting from 2 (Y=R4C(O)Oxe2x80x94) without isolation of intermediary 2 (Z=OMe, X=S, Se). It allows also to use both separated diastereomers of 2 (Y=O, X=S, Se) for synthesis of one diastereomer of the same compound of formula 2 of desired configuration at the P-atom via compound of formula 2 (X=S or Se, Y=R4C(O)Oxe2x80x94).
Within the next variant of the invention, one of the separated diastereomers of 2 (Y=O, X=S, Se) possessing an absolute configuration identical with that desired for the product 1, is alkylated with known alkylating reagents, preferably alkyl halides 8 of general formula R6W, where R6 stands for methyl, cyanomethyl, halogenoacyl, benzyl or aromatic ring substituted benzyl, while W means a chlorine, bromine or iodine atom. The resulting compound of formula 2, where
a) R1 stands for protecting group, preferably 4,4xe2x80x2-dimethoxytrityl (DMT), 9-phenylxanthene-9-ol (Px) or trialkylsilyl group,
b) R2 is a hydrogen atom, protected hydroxyl group, halogen, chloroalkyl, nitrile, azide, protected amine, perfluoroalkyl (containing up to four carbon atoms), perfluoroalkoxyl (containing up to four carbon atoms and up to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl, OQ1, SQ1, NHQ1, where Q1 stands for alkyl (C1-C4), aryl (C6-C12), alkenyl (C3-C12) or alkynyl (C3-C12),
c) B stands for a purine or pyrimidine base (appropriately protected if necessary),
d) Z is selected from Q1 or vinyl, ethynyl, aminomethyl or aminoethyl substituents,
X means oxygen and Y means SR6 or SeR6, where R6 has the above mentioned meaning, is reacted with compound of formula 6, where B stands for a purine or pyrimidine base (appropriately protected if necessary), and Rx is a protecting group, preferably aroyl, acyl, alkoxycarbonyl, or the next unit of elongated oligonucleotide chain. This reaction is catalyzed by strong bases, preferably organic bases as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). An additional activator of this process may be selected from a group consisting of lithium salts, preferably lithium halides, most preferably lithium chloride. The resulting compound of formula 1, where X means an oxygen atom and other substituents have the above mentioned meanings, is isolated using known methods. This product has the absolute configuration identical to that of the product 1 obtained by oxidation of compound of formula 1, where X means a sulfur or selenium atom.
The second diastereomer of of formula 2 (X=S, Se, Y=O) is acylated with compound of formula of formula 7, and then condensed with compound of formula 6 as in the second variant of the process.
The resulting compound of formula 1, where X means a sulfur or selenium atom is isolated using known methods, and oxidized using known oxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, to yield compound 1, where X means an oxygen atom and R1, R2, Rx, B and Z have the above mentioned meanings.
This means, that described above variant of the invention allows, starting from both separated diastereoisomers of 2 (X=S, Se, Y=O) which are independently converted on two different ways (vide supra) to yield one diastereoisomer of the product 1 of desired absolute configuration at the P-atom, where X means an oxygen atom and other substituents have the above mentioned meanings.